Vascular grafts include a wide variety of natural and synthetic tubular structures that may or may not contain valves. Valves in these tubular structures are usually intended to direct the flow of blood (or other nutrient materials) in one direction by preventing the backward flow of this (these) liquid solution(s). Examples of valved tubular structures include aortic, pulmonary, and mitral valves present in the hearts of most vertebrate animals and veins used to return blood flow from the periphery of the body to the heart for recirculation.
Vascular grafts constructed of synthetic materials include devices constructed from man-made polymers, notably Dacron and Teflon in both knitted and woven configurations such as those marketed by W. L. Gore, Inc. and Impra, Inc. where various forms of polytetrafluoroethylene (PTFE) are molded into a wide array of tubule structures (see for example U.S. Pat. Nos. 4,313,231; 4,927,676; and 4,655,769.
Natural vascular grafts, taken in the context of the invention, include valved and non-valved tubular structures obtained by methodologies broadly classified under the term “tissue engineering”. Notably, tissue engineered blood vessels such as described in U.S. Pat. Nos. 4,539,716, 4,546,500, 4,835,102, and blood vessels derived from animal or human donors such as described in U.S. Pat. Nos. 4,776,853, 5,558,875, 5,855,617, 5,843,181, 5,843,180, and a pending patent application entitled “A Process for Decellularizing Soft-Tissue Engineered Medical Implants” (patent application Ser. No. 09/528,371 incorporated herein in its entirety). The present invention involves vascular grafts derived using a novel process associated with tissue engineering as well as a novel bioreactor device for use in the process.
Tissue engineered natural vascular grafts, hereinafter vascular grafts, can be manufactured by processing of natural vascular grafts (including for example, veins, arteries, and heart valves.) with the objective of removing the cellular elements without damaging the matrix structure of that tissue—a “reductionist” approach. This approach is generally referred to as decellularization and is the subject of several patents, of which U.S. Pat. No. 4,801,299 by Brendel and Duhamel is considered as one of the earliest such patents, and pending patent applications as described above. Decellularization of tissues has been attempted by incubating tissues in the presence of detergents, both anionic and nonionic, with and without digestion of nucleic acids.
Tissue engineered natural vascular grafts have also been constructed using a “constructionist” approach. This approach involves the extraction of natural cellular and matrix components to obtain purified (or partially purified) fractions and then using these fractions to reconstruct a vascular graft from individual components. Alternatively, specific components of a vascular graft, for example collagen(s), can be obtained using recombinant DNA technologies and such highly purified and homogeneous materials used in the construction of natural vascular grafts via tissue engineering. Methods and materials for 3-dimensional cultures of mammalian cells are known in the art. See, U.S. Pat. No. 5,266,480. Typically, a scaffold is used in a bioreactor growth chamber to support a 3-dimensional culture. The scaffold can be made of any porous, tissue culture compatible material(s) into which cultured mammalian cells can enter and attach.
Both the reductionist and constructionist approaches are attempts to provide an acellular matrix that can be used directly as an acellular graft.
The invention provides a bioreactor approach to reseeding of vascular grafts, such as a decellularized aortic heart valve. The approach involves removal of the basement membrane by enzymatic digestion. This removal of basement membrane is followed by pressure-differential induced movement of fibroblastic cells in a solution into the matrix structure and reendothelialization by incorporation of endothelial cells into a collagenous/noncollagenous solution. This latter solution is compacted, as necessary, by “pressure” binding of this mixture onto the luminal surface to recreate a “basement membrane” containing endothelial cells. Cells are induced to resume metabolic activities following treatment with specific growth factors, for example fibroblast growth factor, or platelet aggregation under a pulsatile flow of nutrient solutions. The novel design of the bioreactor facilitates the processes described in the present invention.